An ongoing concern in the pharmaceutical and biotechnology industries is the administration of a steady dosage of a therapeutic agent. Conventional delivery methods such as pills or injections typically provide a large spike of drug that decays over a period of time until the next dosage. The main limitation of such a dosing regimen is that the optimum level of drug is only present in the patient for a very brief period of time. Shortly following administration of the drug, it will be present at too high a level. The level will then decay, passing briefly through the optimum point, and remain at a suboptimum level until the next dosage. For many indications, the preferred dosage profile is one of a constant level within the patient--a difficult problem outside a hospital setting. Thus, dosage forms that have a built in time-release mechanism are of strong interest to the pharmaceutical and biotechnology industries. Such formulations have the further advantage of decreased dosage frequency, as the carriers can be designed to release drug for periods of many hours to several months. In consequence, continual patient vigilance is not required, and patient compliance with the prescribed dosage regimen is improved. By maintaining optimal treatment parameters, a reduction in the overall amount of drug administered becomes possible.
In the formulation of controlled release therapeutics, it is frequently desirable to disperse the material into very fine, uniform particles. Such particles may then be advantageously incorporated into controlled-release delivery vehicles such as polymeric microspheres or implantable devices, or delivered to the lungs as an aerosol. With proteinaceous therapeutics, the generation of such fine particles is particularly problematic. Table 1 provides a summary of conventional processing methods and their drawbacks.
TABLE 1 ______________________________________ Conventional Size Reduction Methods for Proteins Method Drawbacks ______________________________________ Mechanical milling Protein denaturation by mechanical shear and/or heat Spray drying Protein denaturation at gas-liquid inter- face and/or heat for solvent vaporization Fluid energy grinding High velocity gas can yield electrostatically charged powders, size reduction inefficient for soft proteinaceous powders Lyophilization Broad size distribution, protein specific appli- cability and protocols Antisolvent precipitation Protein denaturation by organic solvents, solvent removal, solvent residuals, particle size control difficult Salt precipitation Protein specific applicability, salt removal or contamination, particle size control difficult ______________________________________
Over the past decade, it has been demonstrated that compressed gases, liquefied gases, and materials intermediate to gases and liquids known as supercritical fluids can be used to create fine, uniform powders. Materials of this type will generically be referred to as "critical fluids" even though in some cases they would be rigorously defined as a compressed or liquefied gas. Carbon dioxide is a good example of such a critical fluid, with a critical temperature of 31.degree. C. and a critical pressure of 1070 psia. In some instances it is useful to alter the solvation properties of a critical fluid by adding small amounts of what are known as modifiers or entrainers. Examples of entrainers include ethanol, methanol and acetone. The definition of critical fluid is meant to include critical fluids containing modifiers. A critical fluid may also be comprised of a mixture of critical fluids.
A critical fluid displays a wide spectrum of solvation power because its density is strongly dependent on both temperature and pressure--temperature changes of tens of degrees or pressure changes by tens of atmospheres can change solubility by an order of magnitude or more. The readily adjusted solubility of compounds in critical fluids is the basis of a number of precipitation processes that have appeared in the literature. The technique of varying solubility to cause precipitation is limited, however, by the fact that the compound of interest must have significant solubility in the critical fluid for at least some temperature and pressure conditions. Many materials of interest, such as proteins and peptides, are too polar to dissolve to any extent in critical fluids and hence do not fulfill this criterion. It is this limitation that has led to the development of the gas antisolvent (GAS) technique, which name refers to the fact that compressed gases (or liquefied or supercritical gases) are used as antisolvents. In this method, a compressed fluid is added to a conventional organic liquid solvent containing the solute to be crystallized. If compressed gas is used as the antisolvent, its dissolution into the solvent is typically accompanied by a reduction in density and change in polarity of the solvent mixture, and consequently, a reduction of the liquid's solvation power relative to a particular substance. As a result, the mixture becomes supersaturated, which causes crystals to form. If a supercritical fluid or liquefied gas is used as the antisolvent, however, the mixture density may actually be greater than that of the neat solvent. In this case, precipitation is presumably caused primarily by the polarity change of the mixture. In the GAS crystallization process, it is essential that solids have low solubility in the selected compressed fluid.
The GAS technique has been used by a number of workers to produce protein particles. Table 2 provides a survey of this work, which in all cases utilized CO.sub.2 as the antisolvent. In the studies of Yeo et al. and Winters et al. included in Table 2, retention of enzyme activity was monitored. Yeo et al. carried out in vivo testing of the supercritically processed insulin in rats, finding full retention of activity. Winters et al. carried out secondary structure analysis of their supercritically processed proteins using Raman and Fourier transform infrared spectroscopy and found major conformational changes. However, upon redissolution in aqueous solution, the proteins appeared to largely regain their native configuration. The conclusion to be drawn from these studies is that, for at least some proteins, exposure to critical fluids and shearing during decompression does no significant damage to the proteins.
While some success has been achieved with GAS, there is a need for a process that can create small protein particles without the need for dissolution in an organic solvent.
TABLE 2 __________________________________________________________________________ Antisolvent Precipitation of Proteins Using Carbon Dioxide. MW Particle Size Compound kDa Solvent T, .degree. C. P, psia .mu.m Ref. __________________________________________________________________________ Insulin 6 Dimethylsulfoxide 25, 35 1280 0-4 a Insulin 6 N,N-dimethyl formamide 35 1280 0-4 a Insulin 6 10% H.sub.2 O in ethanol 35 1340 0-5 b Catalase 240 10% H.sub.2 O in ethanol 35 1340 1 b Insulin 6 Dimethylsulfoxide 28-46 1350-2115 1-5 c Lysozyme 14 Dimethylsulfoxide 27-45 1093-1713 1-5 c Trypsin 23 Dimethylsulfoxide 27-47 1093-2026 1-5 c __________________________________________________________________________ References: a Yeo, SD, Lim, GB, Debenedetti, P. G. and Bernstein, H., Formation of Microparticulate Protein Powders Using a Supercritical Fluid Antisolvent, Biotechnology and Bioengineering, 41: 341-346, 1993. b Tom, J. W., Lim, G. B., Debenedetti, P. G. and Prud'homme, R. K., Applications of Supercritical Fluids in the Controlled Release of Drugs, ACS Symposium Series 514, American Chemical Society, Washington, D.C., 1993. c Winters, M. A., B. L. Knutson, P. G. Debenedetti, H. G. Sparks, T. M. Przybycien, C. L. Stevenson and S. J. Prestrelski, Precipitation of Proteins in Supercritical Carbon Dioxide J. Pharm. Sci. 85: 586-594, 1996